Breast cancer is the most common cancer among women, other than skin cancer. It is the second leading cause of cancer death in women, after lung cancer. Almost 180,000 women in the United States will be diagnosed with invasive breast cancer in 2007 and over 40,000 women will die from the disease in a year. It has been suggested that the failure of existing therapies may be due to the presence of a subpopulation of cells in the bulk of the tumor that are resistant to radiotherapy and chemotherapy. These cells, called breast cancer stem cells, have self-renewal and multi-pluripotency characteristics. Sonic hedgehog (SHH) signaling plays an important role during normal mammary gland development and it has been recently demonstrated that this signaling pathway is activated in breast carcinoma and that it regulates the behavior of breast cancer stem cells. The SHH pathway therefore constitutes a therapeutic target for the development of new breast cancer therapeutics.
The SHH gene belongs to a human gene family with three genes that encode secreted glycoproteins implicated in multiple developmental processes, including the regulation of cell identity, proliferation and survival (Ingham & McMahon (2001) Genes Dev. 15:3059-3087). SHH is the most widely expressed of the family and the one that has been implicated in human cancer. SHH signals are conveyed intracellularly by the membrane proteins PATCHED (PTCH1) and SMOOTHENED (SMOH). In the absence of SHH, PTCH11 inhibits SMOH, thereby inhibiting the downstream transduction cascade. On binding of SHH to PTCH1, inhibition is released, SMOH signals, and a macromolecular complex that is associated with the cytoskeleton is activated. The reception of the SHH signal in the responding cell results in the activation of target genes through the transcription factors of the GLI family. Three GLI genes have been identified that code for proteins with partially divergent functions (Ruiz i Altaba, et al. (2003) Curr. Opin. Genet. Dev. 13:513-521). In general, GLI1 acts primarily as an activator, GLI2 as both an activator and repressor, and GLI3 mostly as a repressor (Ruiz i Altaba, et al. (2003) supra; Jacob & Briscoe (2003) EMBO Rep. 4:761-765). However, these functions are context-dependent (Bai, et al. (2004) Dev. Cell. 6:103-115; Karlstrom, et al. (2003) Development 130:1549-1564; Persson, et al. (2002) Genes Dev. 16:2865-2878; Ruiz i Altaba (1998) Development 125:2203-2212). SHH-GLI pathway signaling has been shown to be blocked by interaction of a plant-derived alkaloid known as cylopamine with SMOH (Chen, et al. (2002) Genes Dev. 16:2743-2748).
The first link of SHH to cancer was the identification of mutations in the PTCH1 gene in patients with Gorlin's or Basal Cell Nevus Syndrome (Hahn, et al. (1996) Cell 85:841-851; Johnson, et al. (1996) Science 272:1668-1671). These patients develop a variety of tumors at higher frequency and with an earlier onset that normal, including basal cell carcinoma (BCC) of the skin and medulloblastoma of the cerebellum. It has also been shown that SHH signaling is active in the majority of sporadic BCCs (Dahmane, et al. (1997) Nature 389:876-881) and in brain tumors, including medulloblastomas and gliomas (Dahmane, et al. (2001) Development 128:5201-5212; Berman, et al. (2002) Science 297:1559-1561; Clement, et al. (2007) Curr. Biol. 17:165-172). The SHH signaling pathway has also been linked to prostate cancer (Karhadkar, et al. (2004) Nature 431:707-712; Sanchez, et al. (2004) Proc. Natl. Acad. Sci. USA 101:12561-12566; Sheng, et al. (2004) Mol. Cancer. 3:29), small cell lung cancer (Watkins, et al. (2003) Nature 422:313-317), lung adenocarcinoma (Yuan, et al. (2007) Oncogene 26:1046-1055), melanoma (Stecca, et al. (2007) Proc. Natl. Acad. Sci. USA 104:5895-5900), and pancreatic cancer (Berman, et al. (2003) Nature 425:846-851). Importantly, treatment of cancer cells derived form these various types of cancer with cyclopamine in vitro or in vivo induces a decrease in proliferation, an increase of apoptosis, or a decrease of metastasis. The effects of cyclopamine are specific as they are rescued by expression of GLI1, which acts downstream of SMOH in the signaling pathway; further, the effects of cyclopamine are mimicked by inhibition of SMOH by RNA interference (Clement, et al. (2007) Curr. Biol. 17:165-172; Stecca, et al. (2007) Proc. Natl. Acad. Sci. USA 104:5895-5900).
Studies have also linked the SHH signaling pathway with mammary gland function and breast cell proliferation. Studies in transgenic mice, specifically Gli2+/− and Ptc1+/−, have demonstrated a role for SHH signaling in mammary gland development (Lewis, et al. (2001) Dev. Biol. 238:133-144; Lewis, et al. (1999) Development 126:5181-5193), although the precise role of the SHH signaling pathway in this process remains to be elucidated. Moreover, constitutive activation of the SHH pathway in transgenic mice through the expression of activated SMOH in the mammary epithelium results in increased proliferation of progenitors cells and leads to ductal dysplasia (Moraes, et al. (2007) Development 134:1231-1242). In humans, mammary stem cells have active SHH signaling and treatment of mammosphere cultures, generated from mammary stem cells, with recombinant SHH induces an increase in the number of sphere-initiating cells and mammosphere size (Liu, et al. (2006) Cancer Res. 66:6063-6071). Addition of cyclopamine to mammospheres has the inverse effects (Liu, et al. (2006) supra). Malignant mammospheres obtained from human breast cancer samples respond in a similar manner to modulation of SHH signaling (Liu, et al. (2006) supra). Thus, as in the case of gliomas (Clement, et al. (2007) Curr. Opin. Biol. 17:165-172), stem cells in breast cancers appear to require an active SHH pathway for continued proliferation and survival.
There is a need for new therapeutics for treatment of cancer, including breast cancer, and the SHH pathway is an attractive target. Although cyclopamine has been shown to be effective on treatment of cancer using a variety of mouse models and human cells are also sensitive, cyclopamine is difficult to synthesize and its purification from corn lillies (Veratrum californicum.) is expensive. A number of derivatives have been produced, some slightly more active such as KAAD-cyclopamine (Sicklick, et al. (2006) Carcinogenesis 27(4):748-57). Other derivatives of cyclopamine are disclosed, e.g., in U.S. Pat. No. 6,432,970; 6,291,516; 6,552,016; 6,686,388; and 7,230,004 as well as U.S. Patent Application Nos. 2006/0074030, 2004/0060568, 2004/0110663, 2004/0127474, and 2006/0020020; Chen et al. (2002. Proc. Natl. Acad. Sci. USA 99:14071-14076); and Frank-Kamenetsky, et al. (2002) J. Biol. 1:10).
However, there remains a need for new derivatives of cyclopamine that are easier to produce and have greater activity against cancer.